The goal of this project is to define the roles of rRab22b in the formation of myelin and of endocytic compartments. Myelin is a highly specialized multilamellar structure that surrounds axon segments. The integrity of the myelin sheath is essential for normal conduction. Disruptions of the myelin sheath that occur in several diseases, including multiple sclerosis and lysosomal disorders have irreparable consequences. Myelin biogenesis is a highly regulated process that requires coordination of numerous oligodendrocytic events involved in lipid and protein synthesis, intracellular trafficking of membranes, and changes in cell shape. Membrane trafficking is necessary both for delivery of structural myelin components and for movement of molecules that participate in the signaling mechanisms that regulate myelin biogenesis. Both exocytic and endocytic trafficking pathways play major roles in the formation and maintenance of myelin. The coordination .of these processes is required to preserve the structural and functional organization of oligodendrocytes. Rab proteins play a key role in the regulation of membrane trafficking. Our studies indicated that rRab22b, a novel Rab protein that is present in oligodendrocytes, regulates transport from the trans-Golgi network (TGN) to the endosomes. Other evidence suggests that membrane trafficking from the TGN to the endosomes plays essential roles in the biogenesis of endosomes and lysosomes and in the transport of proteins to myelin. We will determine whether Rab22b is involved in the transport of endosomal and lysosomal proteins from the TGN to the endosomes and whether myelin proteins are transported through this pathway before being targeted to myelin. The studies will be performed in living cells including Hela cells, HOG and primary cultures of rat oligodendrocytes. EYFP- tagged rRab22b will be co-expressed with ECFP-tagged endosomal proteins and lysosomal proteins. The organelles containing the EYFP-tagged and ECFP-tagged proteins will be visualized by double fluorescence microscopy. Time-lapse experiments will carry out to visualize the budding from the TGN of vesicles containing the EFYP-tagged and ECFP-tagged proteins. The role of rRab22b in the transport of proteins from the TGN to the endosomes will be confirmed in experiments using rRab22b dominant negative mutant by analysis of how the transport of ECFP chimeric proteins is affected. Similar experiments will carried out expressing rRab22b-EFYP and ECFP-tagged myelin proteins in HOG and oligodendrocytes. The participation of rRab22b in extension of oligodendrocyte processes and formation of membrane will be assessed by expressing rRab22b mutants in oligodendrocyte primary cultures. [unreadable] [unreadable]